Electrophoresis has become an increasingly indispensable tool in biotechnology and related fields. The ability to separate molecules by means of size, shape and charge has added numerous opportunities to identify specific compounds, determine purity, and allow for isolation of a compound in a relatively pure form. A variety of analytical techniques are predicated on the use of electrophoresis for the separation and analysis of the various components of interest that may be present in a particular sample. For example, electrophoresis may be used to identify a compound, where the components of a complex mixture are first separated and then subsequently identified by using markers such as antibodies, DNA probes or the like. Electrophoresis may also be used in the determination of the molecular weights of components in a sample.
Electrophoresis is usually performed in a separation media which provides for separation of the sample components as they migrate through the gel under the influence of an applied electric field. Generally, separation media which have found use in electrophoresis comprise a network of either linear or cross-linked polymers. Although a variety of different cross-linked and linear polymers have been studied for their suitability in electrophoretic applications, the most commonly employed polymers are agarose and cross-linked polyacrylamide.
Agarose gels, which comprise a linear alternating co-polymer of .beta.-D-galactose and 3,6-anhydro-.alpha.-L-galactose in an electrophoresis buffer, have many advantages in electrophoresis. Because they are thermoreversible, i.e. they undergo a transition from a first flowable state to second gel state in response to a change in temperature, agarose gels are easy to prepare. Furthermore, agarose gels have high mechanical strength, providing for ease of manipulation. Another advantage of agarose gels is their ability to separate large molecules, e.g. DNA from 200 bp to about 50 kbp. Despite these advantages, there are disadvantages to the use of agarose gels as an electrophoretic separation medium. One disadvantage of agarose gels is their inability to provide for adequate resolution of smaller sized components. Other disadvantages of agarose gels include the presence of gel impurities that can result in sample contamination, distortions due to electroosmotic flow, and the like.
Crosslinked polyacrylamide gels, which are prepared through polymerization of acrylamide monomer with a cross-linker, provide alternative separation media that overcome some of the problems associated with agarose. Polyacrylamide gels provide for high resolution of small sized sample components, e.g. they are capable of providing high resolution of DNA ranging in size from 6 to 1000 bp in length. Other advantages of cross-linked polyacrylamide gels are that: (1) they are optically transparent, providing for easy identification of separated sample components, (2) they do not bind charged analytes and do not engender electroosmotic flow, and (3) sample components recovered from the gels are extremely pure, as the gels do not contain contaminants, as are found in agarose gels. Unfortunately, since cross-linked polyacrylamide gels must be prepared in situ, their preparation is complicated and poses health risks, as the acrylamide monomers are toxic.
Because of the limitations of the currently employed electrophoretic separation media, for many electrophoretic applications it would be desirable to have a gel which combined the high resolving power, as well as other advantages, of cross-linked polyacrylamide with the thermoreversible nature of agarose.